Western Blotting Reagents Roundup – November 2022

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Reagent Roundup Western Blotting

The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

The role of the epithelial to mesenchymal transition in cancer drug resistance and recurrence

Multicolor near-infrared Western blots and a combined chemiluminescence and NIR blot imaged using Azure 500 Western blot imager
Figure S2 from Ingruber J et al (2022). Interplay between partial EMT and Cisplatin resistance as the drivers for recurrence in HNSCC. Licensed under CC BY 4.0. Multicolor NIR Western blots (panels A,B,D,E) and a combined chemiluminescence and NIR blot (C) were imaged on an Azure C500 imager.

In recent work, Ingruber et al1 hypothesized that head and neck squamous cell carcinoma (HNSCC) cells are in a partial EMT state, able to switch towards epithelial or mesenchymal phenotypes depending on environmental stimuli, and that this switch contributes to their proliferation and resistance to Cisplatin therapy.

As part of this work, the authors carried out chemiluminescent and near-infrared (NIR) fluorescent Western blots to assess levels of EMT protein markers. The authors used Radiance Plus substrate for chemiluminescent Western blots, and fluorescent secondary antibodies for the near-infrared blots. Western blots were then imaged using an Azure c500 imager. The work found that a partial EMT-like pathway appears to contribute to Cisplatin resistance in the cell line used, and that overexpression of an epithelial marker sensitized cells to Cisplatin while reducing a pro-EMT transcription factor. The results suggest future avenues to research and treat drug-resistant cancers.

The epithelial to mesenchymal transition (EMT) is a reversible process in which epithelial cells undergo biochemical changes to adopt a mesenchymal cell phenotype with increased ability to migrate and increased resistance to apoptosis. The EMT can play a role in normal processes such as embryogenesis and wound healing but also contributes to cancer metastasis and tumor cell migration.

Lipid peroxidation in sporadic Alzheimer's disease

Western blot imaged by fluorescence immunoblotting using Azure Sapphire Imager

In a recent publication, Ramsden et al2 propose a new hypothesis for the mechanism behind sporadic Alzheimer’s disease (AD) in which the initiating factor of AD is lipid peroxidation of the apolipoprotein E protein (ApoE) and of the ApoE receptor. AzureRed total protein stain was used to detect total protein before immunoblotting. The blots were blocked with Fluorescent Blot Blocking Buffer and imaged with the Azure Sapphire Biomolecular Imager (Figure 3C and Figure 3D).

The peroxidation is hypothesized to disrupt important processes required for memory formation and maintenance of structural integrity, initiating a cascade that leads to AD. The proposed mechanism differs from the amyloid cascade hypothesis and would have important implications for AD prevention and therapeutics if confirmed. Lipid peroxidation is proposed to occur at the ligand-receptor interface of ApoE and the ApoE receptor where there are amino acid residues predicted to be susceptible to peroxidation.

Because polyunsaturated lipids are transported by ApoE, the ApoE-ApoE receptor interface may create a microenvironment favorable to lipid peroxidation. The hypothesis accounts for several observations about AD including the anatomic areas of the brain known to be affected, the fact that ApoE variants are associated with sporadic AD, that ApoE is enriched in neurite plaque cores, the significance of amyloid plaques and neurofibrillary tangles, and evidence that lipid peroxidation occurs very early in sporadic AD. To test their hypothesis, the authors conducted fluorescence immunoblotting to detect lipid aldehyde-induced crosslinking of ApoE and the ApoE receptor ApoER2.

Based on these in vitro experiments and additional experiments including immunohistochemistry of human brain samples, the authors conclude that their hypothesis is consistent with experimental observations and deserves additional study.

DISCOVER: Azure Sapphire Biomolecular Imager

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A study of the anti-inflammatory effects of LRP1 ligands

Mantuano et al4 used 3 ECL substrates (Radiance, Radiance Q, and Radiance Plus) from Azure Biosystems in their investigation of the anti-inflammatory action of three ligands of LDL receptor protein-1 (LRP1). Chemiluminescent Western blots imaged on the Azure c300 or on film were key to the study as the authors assessed the components required for enzymatically-inactive tissue-type plasminogen activator (El-tPA), activated α2-macroglobulin (a2M), and a soluble derivative of nonpathogenic cellular prion protein (S-PrP) to activate signal transduction in macrophages.

The results found indicate that lipid rafts and the N-methyl-D-aspartic acid (NMDA) receptor are required by all three ligands studied, while LRP1 was not required by two of the ligands when the ligands were present at high concentrations. In addition to the effects on cell signaling, the ligands studied were also shown to prevent lipopolysaccharide (LPS)-induced shedding of LRP1. Since the soluble LRP1 product is pro-inflammatory, blocking this process is another way LRP1 ligands could convey an anti-inflammatory effect. The differences uncovered between the three ligands’ requirements for signal transduction activation might help clarify their effects on macrophages in various states of differentiation 

DISCOVER: Azure 300 Imager

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Find more publications using Azure reagents and imaging systems on our publications list, or contact us directly for assistance with a specific product by using the form on the left.

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SOURCES

  1. Ingruber J, et al. Interplay between partial EMT and Cisplatin resistance as the drivers for recurrence in HNSCC. Biomedicines. 2022;10(10):2482.
  2. Ramsden CE, et al. Lipid peroxidation induced ApoE receptor-ligand disruption as a unifying hypothesis underlying sporadic Alzheimer’s disease in humans. J Alzheimers Dis. 2022;87(3):1251-1290.
  3. Mantuano E et al. The LRP1/CD91 ligands, tissue-type plasminogen activator, a2-macroglobulin, and soluble cellular prion protein have distinct co-receptor requirements for activation of cell-signaling. Sci Rep. 2022;12(1):17594.
  4. Jäntti MA, et al. Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2022;1867(11):159219.

Western Blotting Reagents Roundup – July 2022

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Reagent Roundup Western Blotting

The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

Phosphorylated MED1 links transcription recycling and cancer growth

Aberrant transcription goes hand-in-hand with oncogenesis. Chen et al1 used Chemi Blot Blocking Buffer and Radiance ECL in Western blot experiments investigating transcription recycling in cancer cells. Uncontrolled transcription initiation and elongation are known to be associated with tumor growth but the authors examined whether Pol II recycling, in which RNA polymerase II re-transcribes the same gene rather than being released after transcription is complete, is also associated with cancer. Using a recycling assay developed in their prior publications, the authors demonstrated that Mediator 1 (MED1), when phosphorylated by CDK9, drives Pol II recycling.

Phosphorylation of MED1 increased during prostate cancer progression and inhibiting CDK9 decreased MED1 phosphorylation, Pol II recycling, and prostate tumor growth. The results suggest MED1 phosphorylation and transcription recycling are involved in cancer growth, and MED1 phosphorylation may provide a biomarker to assess therapeutic response of cancers to CDK9 inhibitors.

SHOP: Chemi Blot Blocking BufferRadiance ECL

Aripiprazole Offsets Mutant ATXN3-Induced Motor Dysfunction

Machado-Joseph disease (MJD) is a dominantly inherited progressive ataxia caused by expansion of a CAG repeat in the ataxin-3 gene. Jalles et al2 used Radiance ECL and AzureRed total protein stain, in addition to the Sapphire Biomolecular Imager, in a study investigating how the antipsychotic drug aripiprazole suppresses MJD pathogenesis. In a C elegans model of MJD, the authors found that aripiprazole improved motor performance and this improvement depended on dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors. Identifying the specific targets of aripiprazole may help develop new therapeutics for MJD with fewer side effects.

DISCOVER: Sapphire Biomolecular Imager

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MARK2 regulates directed cell migration

During metastasis, cancer cells migrate by building out the cytoskeleton at the leading edge of the cell and retracting it at the rear. Pasapera et al3 used Azure’s Radiance ECL in a study of cancer cell cytoskeleton polarization. The authors investigated whether the kinase MARK2, known to regulate the microtubule cytoskeleton in other processes, plays a role in the polarization of the cytoskeleton and directed migration of cancer cells. In osteosarcoma cells, Western blot experiments demonstrated that MARK2 promotes stress fiber formation and myosin II activation and mediates inactivation of myosin phosphatase.

The data suggests MARK2 is a major regulator of cell contractility and adhesion that mediates cancer cell motility.

SHOP: Radiance ECL

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli

Alport syndrome is a congenital, progressive glomerular disease that leads to the progressive loss of kidney function. Madison et alused Radiance ECL and TotalStain Q as well as an Azure 600 Imaging System in a study characterizing the glomerular basement membrane (GBM) in a mouse model of Alport syndrome. The investigators found that collagen a1(III) was deposited in the GBM of Alport mice; in wild type mice, collagen a1(III) is found only in the mesangium.

Quantitative Western blotting was carried out using total protein normalization with TotalStain Q staining as the control and the quantitative Westerns confirmed increased levels of collagen a1(III) in the glomeruli of Alport mice. The presence of collagen a1(III) was found to activate DDR1 receptors and lead to changes in gene expression consistent with podocyte injury. Lack of either of the two collagen receptors on podocytes has previously been shown to slow disease progression. The results indicate aberrant collagen-mediated co-receptor signaling through the DDR1 and a2b1 integrin receptors contribute to podocyte injury and renal pathology in Alport syndrome.

DISCOVER: Azure 600 Imaging System

SHOP: Radiance ECLTotalStain Q

Find more publications using Azure reagents and imaging systems on our publications list, or contact us directly for assistance with a specific product by using the form on the left.

Read other blog posts about publications using Azure:

Shop Reagents Mentioned

FREE WESTERN BLOT eBOOK

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

SOURCES

  1. Chen Z, Ye Z, Soccio RE, et al. Phosphorylated MED1 links transcription recycling and cancer growth. Nuc Acids Res. 2022;500(8):4450-4463.
  2. Jalles A, Vieira C, Pereira-Sousa J, et al. Aripiprazole offsets mutant ATXN3-induced motor dysfunction by targeting dopamine D2 and serotonin 1A and 2A receptors in elegans. Biomedicines. 2022;10(2):370.
  3. Pasapera AM, Heissler SM, Eto M, et al. MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Curr Biol. 2022;32(12):2704-2718.
  4. Madison J, Wilhelm K, Meehan DT, et al. Glomerular basement membrane deposition of collagen a1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin a2b J Pathol. 2022; doi: 10.1002/path.5969.

Intranasal Administration of Neutralizing IgA Increases SARS-CoV-2 Infection in a Hamster Model

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COVID-19 Western Blotting

Infectivity of SARS-CoV-2 is associated with high viral loads in the upper respiratory tract. Alpha and gamma immunoglobulins (IgA and IgG) both neutralize viral infections, with IgA significant in mucosal immunity and IgG predominantly affecting blood. Most studies focus on IgG in SARS-CoV-2 infection, but the role of IgA is less understood.

In a preprint shared on ResearchSquare, Zhou et al investigated whether intranasal IgA could protect against SARS-CoV-2 infection in Syrian hamsters. The study is currently under review so the current version is not peer-reviewed or published in a journal. The authors report that neutralizing IgA antibodies, applied intranasally before SARS-CoV-2 challenge, reduced the amount of virus in the lungs but increased viral infectivity in the nasal turbinate of the hamsters.

INTRANASAL ADMINISTRATION OF NEUTRALIZING IGA INCREASES SARS-COV-2 INFECTION IN A HAMSTER MODEL
Figure 5. B8-dIgA1 and B8-dIgA2 enhance SARS-CoV-2 infection via CD209. C. The CD209 or CD299 overexpressed-HEK 293T cells were pre-treated for 6 hours with 10 ng/ml of B8-dIgA1 or B8-dIgA2 or control dIgA1 or control dIgA2 or PBS, respectively, prior to SARS-CoV-2 infection (MOI: 0.05). Two days after infection, SARS-CoV-2 NP expression (green) was quantified by the mean fluorescence intensity (MFI) after anti-NP IF staining. Statistics were generated using student-t tests. *p<0.05; **p<0.01; ***p<0.001.

Methods used

In the course of this work, the authors used the Azure Sapphire Biomolecular Imager in viral neutralization assays and assays of enhanced infection via CD209. For each assay type, cells were grown in 96-well plates and infected with SARS-CoV-2. The media was removed, cells permeabilized and SARS-CoV-2 was detected with an rabbit anti-virus primary antibody followed by a goat anti-rabbit secondary antibody labeled with Alexa Fluor 488. The resulting fluorescent signal was detected on the Sapphire (Figure 5C).

The authors isolated and characterized 18 RBD-specific monoclonal antibodies from four COVID-19 patients. Five of these showed neutralizing activity against SARS-CoV-2. The one with the tightest binding to RBD was tested in the hamster model to see if intraperitoneal administration of the antibody before or soon after viral challenge would affect infection. It did decrease viral loads in the lungs but did not prevent infection in the nasal turbinate.

The group then engineered monomeric and dimeric IgA1 and IgA2 versions of the antibody and tested these in hamsters, either by intraperitoneal injection or nasal administration. The monomeric IgA1 but not IgA2 protected lungs, while neither prevented nasal infection. When dimeric versions of the IgA1 and IgA2 were tested, they were found to decrease infection in the lungs but increase infection and enhance damage in the nasal turbinate.

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Findings

The authors found that the IgA-mediated enhancement did not depend on the ACE2 receptor. Instead, the targets were dendritic cells expressing CD209, a lectin known to be a receptor for secretory IgA.

How SARS-CoV-2 enters host cells

To enter host cells, the receptor binding domain (RBD) of the SARS-CoV-2 spike protein binds to cellular ACE2 receptors. The spike protein is cleaved and the viral and cellular membranes fuse, allowing the virus into the cell. The spike protein is the target of current SARS-CoV-2 vaccines and is the major antigen target of antibodies in infected individuals. Additionally, RBD-specific IgA have been found in COVID-19 patients.

DISCOVER: Azure Sapphire Biomolecular Imager

In addition to fluorescence imaging, the Sapphire Biomolecular Imager provides densitometry, phosphor, multichannel fluorescence, chemiluminescence and white light imaging of blots, gels, tissues, and more.

Western Blotting Reagents Roundup – September 2020

Categories
Multiplex Reagent Roundup Western Blotting

The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

Adipocyte REVERBα dictates adipose tissue expansion during obesity

Chemiluminescent Western blot Azure Biosystems Protein-Free Blot Blocking Buffer showing REVERBα expression in adipose tissue over 24 hours
Figure F. Western blot blocked with Azure Biosystems Protein-Free Blot Blocking Buffer showing REVERBα expression in adipose tissue over 24 hours.

In a recent pre-print, Hunter et al recently demonstrated that REVERBa, a circadian clock component, modulates white adipose tissue metabolism in response to changes in metabolic state. The researchers created a mouse model in which REVERBa was knocked out in only white adipose tissue and compared these mice to mice missing REVERBa in all tissues. Chemiluminescent and fluorescent Western blots were used to confirm protein changes in knock out mice, and to follow circadian changes in protein levels. All Western blot experiments used Azure’s Protein-Free Blocking Buffer to block nitrocellulose membranes. This blocking buffer is a great choice to enhance signal and reduce background for general Western blots and is compatible with all detection chemistries.

SHOP: Protein-free blocking buffer

Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma using a Cell-Penetrating Peptide

Figure 4. Pancreatic cancer cells were treated with 50 μM R9-caPep for up to 48 h. Representative Western blot analysis was performed to analyze both phosphorylated γ-H2AX and unphosphorylated H2AX. Actin was used as the loading control and were detected by ECL prime or AzureSpectra 800 secondary antibodies and imaged on the Azure c600.
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Smith et al identified a novel potential therapeutic approach for pancreatic cancer. In Molecular Therapy Oncolytics, the researchers examined whether a peptide that mimics a part of the proliferating cell nuclear antigen (PCNA) could interfere with PCNA-protein interactions in pancreatic cancer cells. They found that the peptide killed cancer cells by inhibiting DNA replication and DNA repair, resulting in the accumulation of DNA damage. Fluorescent Western blots were used to assess DNA damage by detecting phosphorylated histone H2AX, a biomarker for double stranded DNA breaks. The secondary antibody used was the Azure Spectra 800 Secondary Fluorescent Antibody.

SHOP: Azure Spectra 800 Secondary Fluorescent Antibody.

Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs

Cantu et al identified a surprising new function of vaccinia virus decapping enzymes. The decapping enzymes D9 and D10 are known to promote degradation of viral and cellular mRNAs. In PLOS Pathogens, the researchers compared genome-wide translation efficiency in cells infected with wild type vaccinia virus vs mutant virus lacking functional decapping enzymes. The results indicated that paradoxically, while promoting degradation of mRNAs, the decapping enzymes are also required for selective translation of certain post-replicative viral proteins whose mRNAs have 5’-poly(A) leaders. Translated proteins were detected on chemiluminescent western blots using Azure HRP-conjugated Secondary Antibodies.

SHOP: Azure HRP-conjugated Secondary Antibodies

2‐Oxothiazolidine‐4‐carboxylic acid inhibits vascular calcification via induction of glutathione synthesis

Patel et al are investigating the potential of a cysteine prodrug, 2‐oxothiazolidine‐4‐carboxylic acid (OTC) to inhibit arterial medial calcification. In a recent article published in the Journal of Cell Physiology, the researchers used Azure’s Radiance ECL HRP substrate to detect markers of cell differentiation in cultured vascular smooth muscle cells (VSMC), grown in a calcifying medium with or without OTC. Western blotting revealed that treatment with OTC reversed the increase in osteoblast markers and rescued the drop in glutathione synthesis enzymes observed in calcifying cells, leading the authors to conclude the drug was effective in vitro and might have clinical relevance. The long-lasting signal and high sensitivity of Radiance ECL substrate allows for reliable comparisons of protein levels between samples.

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You can find more publications using Azure reagents and imaging systems on the Azure publications list, or contact Azure for assistance identifying publications using a specific product.

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